Synthetic peptides containing protective epitopes for the treatment and prevention of periodontitis associated with Porphyromonas gingivalis

ABSTRACT

This invention relates to a peptide selected from the group: FLLDADHNTFGSVIPATGPLFTGTASS LYSANFESLIPANADPVVT-TQNIIVTG LYSANFEYLIPANADPVVTTQNIJVTG TNPEPASGKMWIAGDGGNQP RYDDFTFEAGKKYTFTMRRAGMGDGTD DDYVFEAGKKYHFLLLMKKMGSGDGTE TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD NTFGSVIPATGPL PASGKMWIAGDG EAGKKYTFTMRRA EAGKKYHFLMKKM. It also relates to compositions and use of these peptides for treating and testing  Porphyromonas gingialis.

FIELD OF THE INVENTION

This invention relates to an oral composition and an immunogeniccomposition for the suppression of the pathogenic effects of theintra-oral bacterium Porphyromonas gingivalis associated withperiodontal disease. It also relates to diagnostic tests for thepresence of Porphyromonas gingivalis in subgingival plaque samples andspecific antibodies against P. gingivalis antigens in sera. Thecompositions comprise synthetic peptide constructs corresponding toprotective epitopes of the PrtR-PrtK proteinase-adhesion complex ofPorphyromonas gingivalis. The synthetic peptide constructs are useful asimmunogens in vaccine formulations for active immunization and can beused to generate protein-specific and peptide-specific antisera usefulfor passive immunization and as reagents for diagnostic assays.

BACKGROUND OF THE INVENTION

Periodontal diseases are bacterial-associated inflamatory diseases ofthe supporting tissues of the teeth and range from the relatively mildform of gingivitis, the non-specific, reversible inflammation ofgingival tissue to the fore aggressive forms of periodontitis which arecharacterised by the destruction of the tooth's supporting structures.Periodontitis is associated with a subgingival infection of a consortiumof specific Gram-negative bacteria that leads to the destruction of theperiodontium and is a major public health problem. One bacterium thathas attracted considerable interest is Porphyromonas gingivalis as therecovery of this microorganism from adult periodontitis lesions call beup to 50% of the subgingival anaerobically cultivable flora, whereas P.gingivalis is rarely recovered, and then in low numbers from healthysites. A proportional increase in tile level of P. gingivalissubgingival plaque has been associated with an increased severity ofperiodontitis and eradication of the microorganism from the cultivablesubgingival microbial population is accompanied by resolution of tiledisease. The progression of periodontitis lesions has been demonstratedin monkey, rats and once with the subgingival implantation of P.gingivalis. These findings in both animals and humans suggest a majorrole for P. gingivalis in the development of adult periodontitis.

P. gingivalis is a black-pigmented, anaerobic, proteolytic Gram-negativerod that obtains energy from the metabolism of specific amino acids. Themicroorganism has an absolute growth requirement for iron,preferentially in the form of heme or its Fe(III) oxidation producthemin and when grown under conditions of excess hemin is highly virulentin experimental animals. A number of virulence factors have beenimplicated in the pathogenicity of P. gingivalis including the capsule,adhesins, cytotoxins and extracellular hydrolytic enzymes. In order todevelop an efficacious and safe vaccine to prevent P. gingivaliscolonisation it is necessary to identify effective antigens that areinvolved in virulence that have utility as immunogens to generateneutralising antibodies.

We have purified and characterised a multiprotein complex of cysteineproteinases and adhesins which is a major virulence factor forPorphyromonas gingivalis. This complex was biochemically characterisedand disclosed in International Patent Application No. PCT/AU96/00673.The complex consists of a 160 kDa Arg-specific proteinase withC-terminal adhesin domains (designated PrtR) associated with a 163 kDaLys-specific proteinase also with C-terminal adhesin domains (designatedPrtK).

SUMMARY OF THE INVENTION

The present inventors have identified a number of peptides includingepitopes on the adhesins of the PrtR-PrtK complex of cysteineproteinases and adhesins which is a major virulence factor for P.gingivalis. These sequences are set out in Table 1. TABLE 1 Amino acidsequences of peptides including epitopes of the PrtR-PrtK proteincomplex of P. Gingivalis. Desig- PrtR-PrtK Amino acid sequence nationAdhesin [single letter code] EP1 PrtR27 FLLDADHNTFGSVIPATGPLFTGTASS (SEQID NO:1) EP2 PrtR27 LYSANFESLIPANADPVVTTQNIIVTG (SEQ ID NO:2) EP3 PrtK39LYSANFEYLIPANADPVVTTQNIIVTG (SEQ ID NO:3) EP4 PrtR27TNPEPASGKMWIAGDGGNQP (SEQ ID NO:4) EP5 PrtR27RYDDFTFEAGKKYTFTMRRAGMGDCTD (SEQ ID NO:5) EP6 PrtR44DDYVFEACXKKYHFLMKKMGSGDGTE (SEQ ID NO:6) EP7 PrtR27TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKY TITFTMRRAGMGDCTD (SEQ ID NO:7)

Peptides (EP1-EP7) of Table 1 represent sequences of the adhesin domainsof the PrtR and PrtK protein-adhesin complex.

Accordingly, in a first aspect the present invention consists in acomposition for use in raising an immune response against Porphyromonasgingivalis, the composition comprising a suitable adjuvant and/oracceptable carrier or excipient and at least one peptide of not morethan 50 amino acids which peptide includes at least one P. gingivalisepitope, or multimers of said peptide, the at least one P. gingivalisepitope being selected from the epitopes included within a peptideselected from the group consisting of: (SEQ ID NO:1) EP1FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ ID NO:2) EP2LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:3) EP3LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4) EP4 TNPEPASGKMWIAGDGGNQP,(SEQ ID NO:5) EP5 RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ ID NO:6) EP6DDYVFEAGKKYHFLMKKMGSGDGTE, and (SEQ ID NO:7) EP7TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.

Where the composition includes more than one peptide the peptides may bepresent in the composition as individual peptides or in multimericforms. Where multimeric forms are used the multimer may comprisemultiple copies of the same peptide, however, it is preferred that themultimer includes different peptides. Peptide multimers may be preparedas described in PCT/AU98/00076, the entire contents of which areincorporated herein by reference.

In a second aspect of the present invention consists in a peptide, thepeptide having not more than 50 amino acids which peptide includes atleast one P. gingivalis epitope, the P. gingivalis epitope beingselected from the epitopes included in the peptides selected from thegroup consisting of: (SEQ ID NO:1) EP1 FLLDADHNTFGSVIPATGPLFTGTASS (SEQID NO:2) EP2 LYSANFESLIPANADPVVTIQNIIVTG (SEQ ID NO:3) EP3LYSANFEYLIPANADPVVTTQNIIVTG (SEQ ID NO:4) EP4 TNPEPASGKMWIAGDGQNQP (SEQID NO:5) EP5 RYDDFTFEAGKKYTFTMRRAGMGDGTD (SEQ ID NO:6) EF6DDYVFEAGKKYHFLMKKMGSGDGTE (SEQ ID NO:7) EP7TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD

In preferred forms of the present invention the peptide includes atleast one sequence selected from the group consisting of: NTFGSVIPATGPL(SEQ ID NO:8) LYSANFESLIPANADPVVTTQNIIVTG (SEQ ID NO:2)LYSANFEYLIPANADPVVTTQNIIVTG (SEQ ID NO:3) PASCKMWIAGDG (SEQ ID NO:9)EAGKKYTFTMRRA, (SEQ ID NO:10) and EAGKKYHFLMXKM. (SEQ ID NO:11)

In another preferred embodiment of the present invention the peptideincludes at least one sequence selected from the group consisting of:(SEQ ID NO:1) FLLDADHNTFGSVIPATGPLFTGTASS (SEQ ID NO:2)LYSANFESLIPANADPVVTTQNIIVTG (SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG(SEQ ID NO:4) TNPEPASGKMWIAGDGGNQP (SEQ ID NO:5)RYDDFTFEAGKKYTFTMRRAGMGDGTD (SEQ ED NO:6) DDYVFEAGKKYHFLMKKMGSGDGTE, and(SEQ ID NO:7) TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.

In yet another preferred embodiment of the present invention the peptideis selected from the group consisting of: (SEQ ID NO:1)FLLDADHNTFGSVIPATGPLFTGTASS (SEQ ID NO:2) LYSANFESLIPANADPVVTTQNIIVTG(SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG (SEQ ID NO:4)TNPEPASGKMWIAGDGGNQP (SEQ ID NO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD (SEQ IDNO:6) DDYVFEAGKKYHFLMKKMGSGDGTE, and (SEQ ID NO:7)TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.

As will be readily apparent to persons skilled in this area thesepeptides may be used as antigens in diagnostic tests or as immunogens informulations.

In a third aspect the present invention consists in an antibodypreparation comprising antibodies specifically directed against thecomposition of the first aspect of the invention or the peptides of thesecond aspect of the invention. The antibodies may be either polyclonalor monoclonal antibodies.

As will be readily apparent to persons skilled in this area theseantibodies may be used in diagnostic tests or in pharmaceuticalformulations.

In a fourth aspect the present invention consists in a method ofreducing the prospect of P. gingivalis infection in an individual and/orseverity of disease, the method comprising administering to theindividual an amount of the composition of the first aspect effective toinduce an immune response in the individual directed against P.Gingivalis.

In a fifth aspect the present invention consists in a method of reducingthe prospect of P. gingivalis infection in an individual and/or severityof disease, the method comprising administering to the individual aneffective amount of an antibody of the third aspect.

Peptides can be synthesized using one of the several methods of peptidesynthesis known in the art including standard solid phase peptidesynthesis using-t-butyloxycarbonyl amino acids (Mitchell et al., 1978,J. Org. Chem. 43:2845-2852) using 9-fluorenylmethyloxycarbonyl (Fmoc)amino acids on a polyamide support (Druland et al. 1986, J. Chem. Soc.Perkin Trans. 1 125-137)-by pepscan synthesis (Geysen et al., 1987, J.Immunol Methods 03:259; 1984. Proc. Natl. Acad. Sci. USA, 81:3998) or bystandard liquid phase synthesis.

A variety of methods for the synthesis of multivalent/multipeptide highmolecular weight peptide molecules can be used to synthesize the peptideantigens. This will be achieved using known in the art and novelligation strategies.

Peptides from Table 1 can be synthesized in such a way as to-contain twoligands, which can be the same or different, which may or may not be thecomplementary ligand. These bi-modal peptides can incorporate any ligandthus linkages such as thioether, thioester, hydrazone, oxime,thiazolidine can be utilised for the synthesis of multipeptideconstructs Shao and Tam., 1995, J. Am. Chem. Soc. 117, 3893-3899. Rose,et al 1996, Bioconjugate Chem. 7(5):552-556, Rose. K., 1994. J. Am.Chem. Soc. 116:30-33, Canne., et al 1995, J. Am. Chem. Soc.117:2998-3007, Lu., et al. 1991. Mol. Immunol 28(6):623-630, Liu andTam., 1994, Proc. Natl. Acad. Sci. 91:6584-6588. A novel ligatingstrategy is to use the klown reaction between thioanisole and acryloylpeptides (O'Brien-Simpson et al., 1997, J. Am. Chem. Soc. 119 (6) whichresults in the para substitution of thioanisole by the double bond inacidic conditions. By synthesising and mixing acryloyl-peptides andphenylthio acetyl peptides and exposing them to acidic conditionsligation can proceed by Friedal-Craft allylation. Ligation can beaccomplished between peptides and on to an oligolysine supportderinvatised with one of the ligands. Conditions for ligation canconsist of: Friedal-Craft reaction conditions which are known in the artand known peptide cleavage conditions.

The introduction of ligand groups to form bi-modal peptides can beachieved by coupling a ligand on to free amino groups, which is known inthe art, at the N- or C-terminus of a peptide or within the peptidesequence. This can be achieved by coupling eg. Fmoc(Fmoc) 2,3 diaminopropionic acid or Fmoc Lys (Fmoc)-OH or orthogonally protected lysineresidues such as Fmoc Lys (Mtt)-OH using standard peptide couplingprotocols on to the N-terminus or introduced at the C-terminus or withinthe peptide sequence. After deprotection, ligand groups can be coupledon to the amino groups and by selective deprotection of eg. Fmoc Lys(Mtt) different ligands can be coupled on to a single peptide. At anypoint in the synthesis spacer moieties can be introduced between thepeptide and the ligands and/or between the ligands, which may be used toreduce steric hindrance in the ligation reaction. FIG. 1 shows thesynthesis protocol.

Peptide ligation can be achieved in solution or on the solid phase. Theincorporation of different ligands and selective protection of oneligand can allow the synthesis of multivalent, multipeptide constructs,where by, peptides are ligated sequentially. This strategy has theadvantage that the orientation and order of peptides ligated is knownand can be controlled. Protecting groups for ligands can be for exampleFmoc, allyloxycarbonyl (Aloc) or nitrocinnamyloxycarbonyl (Noc) whichare stable to standard cleavage conditions but are easily removed underbasic conditions or catalytic allyl transfer. FIG. 2 shows the ligationscheme for the synthesis of multivalent peptide constructs usingbi-modal peptides. The protocol can be adapted for a variety of ligationchemistries by simply altering the ligands which are coupled to thepeptide to form the bi-nodal peptide.

The step wise addition of each peptide can be achieved on the solidphase. This can be achieved by synthesising a peptide on to the solidsupport via a base labile handle ec. 4-hydroxymethyl benzoic acid. Thiscan allow full side chain deprotection of the peptide with the peptideremaining attached to the solid support. This would allow ligation tostill be carried out in aqueous solvents similar to those used forsolution phase ligation except that separation of the ligand productfrom unreacted bi-modal peptide can be achieved by simply washing thesolid support. The reaction can be monitored by ninhydrin ortrinitrobenzene sulphonic acid tests, where by, lysine residues withinthe bi-modal peptide would need to be protected eg. with(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) which is stable toacid cleavage but can be removed with hydrazine. FIG. 3 shows theligation strategy for the solid phase.

Bi-modal peptides can be synthesized so that ligands are at the N- andC-terminus. This would allow the preparation of cyclic peptides and theformation of di-peptide constructs where by peptides can run parallel oranti parallel to each other by either coupling N- to N- and C- toC-termini or N- to C-termini together respectively (FIG. 4).

Another technique for the synthesis of multivalent peptide constructs isto ligate peptides on to an oligolysine support (Rose, et al 1996,Bioconjugate Chem. 7(5):552-556, Canne., et al 1995, J. Am. Chem. Soc.117:2998-3007 and Lu., et al, 1991, Mol. Immunol 28(6);623-630). Byincorporating a number of different ligands and or protected ligands onto the lysine support, peptides can be ligated to a particular positionon the support. Ligation chemistries such as oxime or hydrazone withhaloacylation and Friedal-Craft alkylation can be used sequentiallywithout the need for ligand protection. Ligand protection can be used toincrease the number of different peptides incorporated on to the lysinesupport. FIG. 5 demonstrates the synthesis protocol.

Another method known in the art is the synthesis of acryloyl peptidesand their polymerisation with acrylamide (O'Brien-Simpson et al., 1997,J. Am. Chem. Soc. 119 (6)) or acryloyl amino acids. Peptides from thePrtR-PrtK protein complex listed in Table 1 can be acryloylated andpolymerised either singularly or in combination. Although this methodallows the polymerisation of a number of peptides together the order inwhich peptides are incorporated can not be controlled.

The final peptide construct may or may not contain all, sum or part ofthe peptides listed in Table 1. Also the construct may or may notcontain promiscuous T-cell epitopes known in the art (Kaumaya et al1994, in Solid Phase Synthesis, Ed Epton, R) or a derived sequence fromstructural/binding motifs of WIC class II binding peptides (O'Sullivanet al., 1991, J. Immunol, 147:2663-2669, Hammer et al., 1993, Cell,74:197-203 and Alexander et al., 1994, Immunity, 1:751-761).Furthermore, lipid moieties such as palmitic acid or cholesterol can beincluded to enhance the immunogenic properties of the peptide construct.Enzymatic cleavable sequences known in the art (Duncan et al., ref) orderived sequences from cleavage motifs (Van Noort and van der Drift.,ref) can also be incorporated with the peptide construct.

The synthetic peptide antigens identified in Table 1 are of particularinterest for diagnostics and neutralisation by passive immunity throughoral compositions containing neutralising antibodies and by vaccinedevelopment. The superiority of these synthetic peptide antigens toprior disclosed P. gingivalis antigens, is that these peptides have beendemonstrated to react with protective sera from animals and humans. Thepeptides represent sequences in the adhesin domains of the PrtR and PrtKwhich make them ideal for the development of diagnostic andimmunoprophylactic products.

Antibodies against the antigens can be used in oral compositions such astoothpaste and mouthwash to neutralise the antigens and thus preventdisease. Antigen-specific antibodies can also be used for the earlydetection of P. gingivalis in subgingival plaque samples by a diagnosticassay. A vaccine based on these antigens and suitable adjuvant deliveredby nasal spray, orally or by injection to produce a specific immuneresponse against these antigens thereby reducing colonisation andvirulence of P. gingivalis and thereby preventing disease. The peptideantigens of the present invention may be used as immunogens inprophylactic and/or therapeutic vaccine formulations; or as an antigenin diagnostic immunoassays directed, to detection of P. gingivalisinfection by measuring an increase in serum titer of P.gingivalis-specific antibody. Also the synthetic peptides of the presentinvention may be used to generate antigen-specific antibody which may beuseful for passive immunization and as reagents for diagnostic assaysdirected to detecting the presence of P. gingivalis in clinicalspecimens such as subgingival-plaque samples.

As mentioned it is preferred that the composition includes an adjuvant.As will be understood an “adjuvant” means a composition comprised of oneor move substances that enhances the immunogenicity and efficacy of avaccine composition. Non-limiting examples of suitable adjuvants includesqualane and squalene (or other oils of animal origin); blockcopolymers; detergents such as Tween®-80; Quil® A, mineral oils such asDrakeol or Marcol, vegetable oils such as peanut oil:Corynebacterium-derived adjuvants such as Corynebacterium parvum;Propionibacterium-derived adjuvants such as Propionibacterium acne;Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukinssuch as interleukin-2 and interleukin 12; monokines such as interleukin1; tumour necrosis factor; interferons such as gamma interferon;combinations such as saponin-aluminium hydroxide or Quil-A aluminiumhydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract;synthetic glycopeptides such as murarmyl dipeptides or otherderivatives; Avridine; Lipid A derivatives: dextran sulfate:DEAE-Dextran or with aluminium phosphate; carboxypolymethylene such asCarbopol EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g.U.S. Pat. No. 5,047,238); vaccinia or animal poxvirus proteins;sub-viral particle adjuvants such as cholera toxin, or mixtures thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Synthesis of Bi-modal Peptides Although a specific example isshown here any ligand can be introduced at the a or e amino groups oflysine. (a) acylation e.g. amino acid:HOBt:HBTU:DIPEA 1:1:1:1.5 indimethyl formamide (DMF). (b) Fmoc deprotection e.g. 20% piperidine inDMF. (c) Levulinic acid: diisopropyl carbodiimide (DIC) 2:1 indichloromethane (DCM), 1 h. (d) Mtt removal, 3×1% TFA in DCM, 3 mins.(e) Fmoc-Hydrazino benzoic acid: DIC 2:1, in DCM, 1 h. (f) Acid cleavagee.g. TFA: water 95:5.

FIG. 2: Synthesis of multivalent peptide constructs using bi-modalpeptides. (a) Ligation. 5 M urea and 0.1 M NaH₂PO₄ (pH range 3-4.7).Ligation can be monitored by reverse phase analytical HPLC and massspectrometry. (b) Deprotection, e.g. Aloc is removed bypalladium(0)-catalyzed allyl-group transfer to a basic receptor. Theligation product can be purified by preparative HPLC and lyophilised.(c) Ligation. Similar conditions as described in (a). Different ligationchemistries can be used by synthesising peptides with different ligandsand synthesising non-complementary ligands on to the same peptide,thereby avoiding proected ligands. The square symbol indicatesprotection, (L) ligand, (P) peptide.

FIG. 3: Synthesis of multivalent peptide constructs using bi-modalpeptides by solid phase. (a) Deprotection and ligation. The S-acetylprotecting group is removed by aqueous hydroxyamine 0.03 M, pH 7.3.After washing the first peptide can be ligated on to the SH group, 6 Maqueous guanidine hydrochloride and 0-05 M EDTA pH 6.4-6.5 adjusted by 1M Tris HCl under nitrogen. Ligation buffer can contain organic solventssuch as acetonitrile. (b) Deprotection, the S-acetyl protecting groupcan be removed by aqueous hydroxyamine 0.05 M, pH 7.3. (c) Ligation, asdescribed in (a) although different ligation chemistries can be used bysynthesising peptides with different ligands and synthesisingnon-complementary ligands on to the same peptide thereby avoidingproected ligands. The square symbol indicates protection, (L) ligand.(P) peptide, (B) base labile handle, 4 hydroxymethyl benzoic acid.

FIG. 4: Cyclization using bi-modal peptides. (a) Deprotection andcyclisation. Synthesis of bi-modal peptides which have complimentaryligands at their N- and C-termini allows the cyclisation of thesepeptides in aqueous buffers. (i) Ligation. (ii) Deprotection andligation. (iii) Cleavage of the cyclic peptide from the base labilehandle. Example; The peptides shown are from Table 1 and represent majorprotective epitopes on PrtR 27 or PrtK39. (a) Ligation. 95% aqueous TFA.Ligation can be monitored by reverse phase analytical HPLC and massspectrometry. Ligation conditions can be varied to included scavangerscommonly used in peptide synthesis and different acidic conditions toenhance the Friedal-Craft allylation. (b) Deprotection and ligation. TheS-acetyl protecting group can removed by aqueous hydroxyamine 0.05 M, pH7.3. Ligation, 5 M aqueous guanidine hydrochloride and 0.05 M EDTA pH6.4-6.5 adjusted by 1 M Tris.HCl under nitrogen. The ligation straegycan also be accomplished on the solid phase. By selecting which ligandto introduce at the N- and C-terminal parallel and anti-parallel cyclicpeptides can be synthesised.

FIG. 5: Synthesis of multivalent multiple antigenic peptides (MAPs)using alternate ligation chemistries. By using different ligationstrategies a vareity of peptides can be ligated onto a single multipleantigenic peptide. The example shown is of peptides listed in Table I.(a) Ligation, 95% aqueous TFA. Ligation can be monitored by reversephase analytical HPLC and mass spectrometry. Deprotection, Aloc canremoved by palladium(0)-catalyzed allyl group transfer to abasic-receptor after purification the second peptide can be ligated onto the MAP, (c) 8 M urea and 0.1 M NaH₂PO₄ (pH range 3-4.7).

FIG. 6: Serum IgG antibody responses assessed by ELISA to Porphyromonasgingivalis PrtR-27 overlapping peptides. Twenty one PIN-bound peptideswere probed with normal mouse sera (▪), protective mouse sera (□),normal human serum

patient D24 sera

patient H10 sera (▪) and patient D20 sera

ELISAs were developed as per Example 1.

FIG. 7: Maximum lesion size of mice immunised with peptide-DTconjugates. BALB/c: mice were immunised (s.c) with the peptide-DTconjugates (50 μg) administered in IFA for the primary and secondarydoses and challenged (s.c.) 12 days after the second dose with P.gingivalis strain ATCC 332777 (7.5×10⁸ viable cells). Animals weremonitored over a 14 day period for weight loss and lesion size. Data wasanalysed using Kruskell-Wallis rank sum test and Mann-Whitney U-Wilcoxonrank sum test with a Bonferroni correction. *=P≦0.005.ns=notsignificant.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to an oral composition and a vaccine for thesuppression of the pathogenic effects of the intra-oral bacteriumPorphyromonas gingivalis associated with periodontal disease. It alsorelates to diagnostic tests for the presence of Porphyromonas gingivalisin subgingival plaque samples and specific anti-P. gingivalis antibodiesin sera. The peptide antigens of Table 1 can be synthesized individuallyor as multimetric or multipeptide constructs.

The synthetic peptide antigens are used to generate polyclonal ormonoclonal antibodies using standard techniques. The animals used forantibody generation can be mice, rabbits, goats, chickens, sheep,horses, cows etc. When a high antibody titre against the antigens isdetected by immunoassay the animals are bled or eggs or milk arecollected and the serum prepared and/or antibody purified using standardtechniques or monoclonal antibodies produced by fusing spleen cells withmyeloma cells using standard techniques. The antibody (immunoglobulinfraction) may be separated from the culture or ascites fluid, serum,milk or egg by saltine our, gel filtration, ion exchange and/or affinitychromatography, and the like, with salting out being preferred. In thesalting out method the antiserum or the milk is saturated with ammoniumsulphate to produce a precipitate, followed by dialyzing the precipitateagainst physiological saline to obtain the purified immunoglobulinfraction with the specific antibody. The preferred antibody is obtainedfrom the equine antiserum and the bovine antiserum and milk. In thisinvention the antibody contained in the antiserum and milk obtained byimmunising the animal with the antigens is blended into the oralcomposition. In this case the antiserum and milk as well as the antibodyseparated and purified from the antiserum and milk may be used. Each ofthese materials may be used alone or in combination of two or more.Antibodies can be used in oral compositions such as toothpaste andmouthwash to neutralise P. gingivalis and thus prevent disease. Theantibodies can also be used for the early detection of P. gingivalis insubgingival plaque samples by a chairside Enzyme Linked ImmunosorbentAssay (ELISA).

For oral compositions it is preferred that the amount of the aboveantibodies administered is 0.0001-50 g/kg/day and that the content ofthe above antibodies is 0.0002-10% by weight preferably 0.002-5% byweight of the composition. The oral composition of this invention whichcontains the above-mentioned serum or milk antibody may be prepared andused in various forms applicable to the mouth such as dentifriceincluding toothpastes, toothpowders and liquid dentifrices, mouthwashes,troches, chewing gums, dental pastes, gingival massage creams, gargletablets, dairy products and other foodstuffs. The oral compositionaccording to this invention may further include additional well knowningredients depending on the type and form of a particular oralcomposition.

In certain highly preferred forms of the invention the oral compositionmay be substantially liquid in character, such as a mouthwash or rinse.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humectant as described below. Generally, theweight ratio of water to alcohol is in the range of from about 1:1 toabout 20:1. The total amount of water-alcohol mixture in this type ofpreparation is typically in the range of from about 70 to about 99.9% byweight of the preparation. The alcohol is typically ethanol orisopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 4.5 to about 9 and typically fromabout 5.5 to 8. The pH is preferably in the range of from about 6 toabout 8.0, preferably 7.4. The pH can be controlled with acid (e.g.citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered(as with sodium citrate, benzoate, carbonate, or bicarbonate; disodiumhydrogen phosphate, sodium dihydrogen phosphate, etc).

Other desirable forms of this invention, the oral composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a dentifrice, that is a toothpaste (dental cream) or geldentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material. Examples ofpolishing materials are water-insoluable sodium metaphosphate, potassiummetaphosphate, tricalcium phosphate, dihydrated calcium phosphate,anhydrous dicalcium phosphate, calcium pyrophosphate, magnesiumorthophosphate, trimagnesium phosphate, calcium carbonate, hydratedalumina, calcined alumina, aluminum silicate, zirconium silicate,silica, bentonite, and mixtures thereof. Other suitable polishingmaterial include the particulate thermosetting resins such as melamine-,phenolic, and urea-formaldehydes, and cross-linked polyepoxides andpolyesters. Preferred polishing materials include crystalline silicahaving particle sized of up to about 5 microns, a mean particle size ofup to about 1.1 microns, and a surface area of up to about 50,000cm²/gm., silica gel or colloidal silica, and complex amorphous alkalimetal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metalalumino-silicate complexes are particularly useful since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner asillustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4thEdition, pp 510-511. The forms of insoluble sodium metaphosphate knownas Madrell's salt and Kurrol's salt are further examples of suitablematerials. These metaphosphate salts exhibit only a minute solubility inwater, and therefore are commonly referred to as insolublemetaphosphates (IMP). There is present therein a minor amount of solublephosphate material as impurities, usually a few percent such as up to 4%by weight. The amount of soluble phosphate material, which is believedto include a soluble sodium trimetaphosphate in the case of insolublemetaphosphate, may be reduced or eliminated by washing with water ifdesired. The insoluble alkali metal metaphosphate is typically employedin powder form of a particle size such that no more than 1% of thematerial is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight. Other polishingmaterials are typically present in amount of about 30-75% by weight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol, andpolypropylene glycol exemplify suitable humectants/carriers. Alsoadvantageous are liquid mixtures of water, glycerine and sorbitol. Inclear gels where the refractive index is an important consideration,about 2.5-30% w/w of water, 0 to about 70% w/w of glycerine and about20-80% w/w of sorbitol are preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O. 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density or 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellulose and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationsare to be sold or otherwise distributed in suitable labelled packages.Thus a jar of mouthrinse will have a label describing it, in substance,as a mouthrinse or mouthwash and having directions for its use; and atoothpaste, cream or gel will usually be in a collapsible tube,typically aluminium, lined lead or plastic, or other squeeze, pump orpressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature which does notdenature the antibody of the invention, and it is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties while not denaturing theantibody. Suitable examples of anionic surfactants are water-solublesalts of higher fatty acid monoglyceride monosulfates, such as thesodium salt of the monosulfated monoglyceride of hydrogenated coconutoil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate,alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higheralkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propanesulfonate, and the substantially saturated higher aliphatic acyl anodesof lower aliphatic amino carboxylic acid compounds, such as those having12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and thelike. Examples of the last mentioned amides are N-lauroyl sarcosine, andthe sodium, potassium, and ethanolamine salts of N-lauroyl. N-myristoyl,or N-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants suitable for use earth antibodies are condensation productsof ethylene oxide with various reactive hydrogen-containing compoundsreactive therewith having long hydrophobic chains (e.g. aliphatic chainsof about 12 to 20 carbon atoms), which condensation products(“ethoxamers”) contain hydrophilic polyoxyethylene moieties, such ascondensation products of poly (ethylene oxide) with fatty acids, fattyalcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate)and polypropyleneoxide (e.g. Pluronic materials).

Surface active agent is typically present in amount of about 0.1-5% byweight. It is noteworthy, that the surface active agent may assist inthe dissolving of the antibody of the invention and thereby diminish theamount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavoring or sweetening material may also be employed.Examples of suitable flavoring constituents are flavoring oils, e.g. oilof spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavor and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 5.5 to about 8, preferably about 6 to 8, for at least 2weeks up to 8 weeks or more up to a lifetime.

The compositions of this invention can be incorporated in lozenges, orin chewing gum or other products, e.g. by stirring into a warm gum baseor coating the outer surface of a gum base, illustrative of which may bementioned jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticizers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like.

Another important form of the invention is a vaccine based on thesynthetic peptide antigens and suitable adjuvant delivered by nasalspray, orally or by injection to produce a specific immune responseagainst the antigen thereby reducing colonisation of P. gingivalis andreducing virulence thereby preventing disease. Unlike whole P.gingivalis cells or other previously prepared antigens, the peptideantigens described herein are safe and effective antigens for thepreparation of a vaccine for the prevention of P. gingivalis-associatedperiodontal disease. Additionally, according to the present invention,antigenic peptide produced may be used to generate P. gingivalisantisera useful for passive immunization against periodontal disease andinfections caused by P. gingivalis.

The following examples are further illustrative of the nature of thepresent invention, but it is understood that the invention is notlimited thereto. All amounts and proportions referred to herein and inthe appended claims are by weight unless otherwise indicated.

EXAMPLE 1 (i) Identification of Protective Epitopes in the PrtR-PrtKProteinase-Adhesin Complex

The PrtR-PrtK proteinase-adhesin complex was purified as describedpreviously in International Patent Application No. PCT/AU96/00673 andwas shown to confer protection to mice against challenge with P.gingivalis when used as a vaccine. The PrtR-PrtK complex was tested inthe mouse abscess model This model is loosely based on the methods ofKesavalu et al. (1992) [Infect Immun 60: 1455-1464]. A typicalexperiment is outlined below. Briefly BALB/c mice were obtained from ARC(Perth, Australia) and were immunised subcutaneously in the scruff ofthe neck with the preparations and doses according to Table 2 beforechallenge with live P. gingivalis strain W50, which was given at 10weeks of age. Mice were given 2 doses of vaccine at 4 and 1 weeks beforechallenge. Formalin killed P. gingivalis W50 cells were prepared byincubating an aliquot of cells in 0.5% (vol/vol) of buffered formolsaline overnight at 4° C. All preparations were emulsified with an equalvolume of Freund's Incomplete Adjuvant (FIA, Sigma) prior to injection.Animals were bled before and 1 week after the immunisation schedule.Sera were screened by ELISA and using a P. gingivalis sonicate as theadsorbed antigen. TABLE 2 Immunization schedule No. of Group DosesTreatment n 1 2 1 × −10⁹ Formalin killed P. gingivalis cells 11 in FIA¹2 2 Affinity purified P. gingivalis PrtR-PrtK complex  5 in FIA 3 2Tris-cysteine buffer in FIA 10 4 2 Tris-cysteine buffer 10¹FIA = Freund's incomplete adjuvant

For the preparation of the bacterial challenge P. gingivalis cells weregrown at 37° C. on lysed horse blood agar (HBA) plates until day 3 or 4in an anaerobic chamber (Mark 3 Anaerobic Workstation, Don WhitleyScientific Limited; with an air mixture of 8% H₂, 12% CO₂, 80% N₂), thenpassaged into 20 ml of brain heart infusion broth (BHIB: Oxoid)supplemented with 0.5 g/L cysteine and 1 mg/L haemin for 24 hours in astandard incubator at 37° C. Finally, 3 ml of this culture was added to400 ml of BHIB-cysteine media and incubated for approximately 15 hoursin a standard incubator at 37° C. until the optical density at 650 nmreached 0.18. The cells were then pelleted by centrifugation at 10,000 gfor 30 minutes using a JA10 rotor in a Beckman High Speed centrifuge andthen resuspended to a final dilution of 3×10¹⁰ cells per ml inBHIB-cysteine media according to previously established growth curvesfor the W50 strain used in these experiments. Mice were marked foridentification, their backs and chests shaved to make measurement oflesions possible, then weighed prior to inoculation with the challengedose at a single site in the middle of the back. A 0.1 ml dose was givenrepresenting a predicted challenge dose of 3×10⁹ bacteria per mouse. Theinoculum dose was confirmed by culturing various dilutions of thechallenge dose on lysed HBA plates and examining the number of colonies7 days later.

Following challenge mice were examined daily for the number and size oflesions on their body and their size estimated by measuring theapproximate surface area in mm² involved. Previous experiments had shownthat in unimmunized mice, lesions developed on the belly of the micefollowing inoculation of live bacteria into the back or side. Anydistressed animals were culled. Observations were carried out over twoweeks and a summary of one such experiment is summarised below in Table3. In this experiment while a dose of 3×10⁹ bacteria per mouse was thedesired number of bacteria, after plating out of the inoculum it wascalculated that each mouse actually received a challenge dose of3.17×10⁹ live P. gingivalis-bacteria strain W50.

When mice were immunised significant reductions (p<0.005) were seen inthe size of the lesions with whole formalin killed P. gingivalis strainW50 cells (Group 1), and the PrtR-PrtK complex (Group 2) when comparedwith the lesion size of the animals receiving FHA (Group 3) (Table 3).These results clearly show that the. PrtR-PrtK complex works effectivelyas an immunogen. The only group of animals that had a number of animals(40%) that exhibited no visible lesions at all was the PrtR-PrtK complexgroup (Group 2). All other groups, including formalin killed cells(Group 1), had all animals exhibiting visible lesions indicating thatthe PrtR-PrtK complex was a better immunogen than formalin killed cells.TABLE 3 Immunisation with the PrtR-PrtK complex can protect mice fromchallenge with P. gingivalis Lesion size Mean maximum lesion size Groupmm² p* 1  30.2 ± 28.4^(†) 0.0008 2  30.0 ± 36.0 0.0028 3  86.8 ± 41.1 —4 201.7 ± 125.8 0.012*probability calculated by Mann Whitney rank sum test comparing Group 3with other groups.^(†)mean ± SD

The protective sera from Group 2 mice immunized with the PrtR-PrtKcomplex were collected and pooled and used in a Western Blot with thePrtR-PrtK immunogen. As well as the mice protective sera a Western blotwas also carried out using sera from two patients with periodontitis(D24, D20) and a healthy patient (H10) with no signs of periodontitisbut who harboured P. gingivalis in sub-gingival plaque. Patient D20 hadsevere advanced periodontitis whereas patient D24 had only low levels ofdisease.

Immunoblotting

Purified PrtR-PrtK complex was subjected to SDS-PAGE using 12.5% oacrylamide gels (1 mm) in a mini-gel system (BioRad. Richmond. Calif.).Proteins were electrophorectically transferred onto PVDF membrane. Aftersectioning of the membrane the molecular weight standards were stainedwith 0.1% w/v CBB R250. The remaining sections were blocked for 1 hourat 20° C. with 5% w/v non-fat skim milk powder in TN buffer (50 mMTris-HCl, pH 7.4, 100 mM NaCl). Sections were subsequently incubatedwith the following antisera diluted 1:25 with TN buffer, patient H10,patient D24, patient D20 or mouse protective anti PrtR-PrtK sera. Afterfive hours at 20° C. the sections were washed (4×TN buffer containing0.05% v/v Tween 20) and then incubated for an hour at 20° C. with theappropriate conjugate antibody; anti-human IgG horse radish peroxidaseconjugate or anti-mouse IgC horse radish peroxidase conjugate. Afterwashing (4×TN buffer containing 0.05% v/v Tween 20) bound antibody wasdetected with 2.8 M 4-chloro-1-napthol in TN buffer containing 16.6% v/vmethanol and 0.05% v/v of a 30% H₂O₂ solution. Colour development wasstopped by rinsing the membranes with Mlilli Q water.

A protein band at 44 kDa was shown to react with all of the sera tested(data not showed. The protective mouse sera and sera from patient H10who does not have periodontitis also bound to a protein band of 27 kDa(PrtR27 adhesin). Patient sera from D20 (advanced periodontitis) did notreact with this 27 kDa protein suggesting that antibodies directedtoward the 27 kDa adhesin may have provided protection againstperidontitis in patient H10 and the immunoprotected mice.

Epitope Mapping Analysis

Twenty overlapping 13mer peptides (overlay by 6 and offset by 7residues) corresponding to the N-terminal 148 residues of the PrtR27were synthesised by Chiron Technologies (Melbourne, Australia) using themultipin peptide synthesis system. The sequence of the N-terminal 148residues of the PrtR27 adhesin is as follows: (SEQ ID NO: 12)ANEAKVVLAADNVWDGNTGYQFLLDADHNTFGSVIPATGPLFTGTASSDLYSANFESLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPA.

The overlapping peptides synthesised were: ANEAKVVLAADNV (SEQ ID NO:13)LAADNVWDGNTGY (SEQ ID NO:14) DGNTGYQFLLDAD (SEQ ID NO:15) FLLDADHNTFGSV(SEQ ID NO:16) NTFGSVIPATGPL (SEQ ID NO:17) PATGPLFTGTASS (SEQ ID NO:15)TGTASSDLYSANF (SEQ ID NO:19) LYSANFESLIPAN (SEQ ID NO:20) SLIPANADPVVTT(SEQ ID NO:21) DPVVTTQNIIVTG (SEQ ID NO:22) NIIVTGQGEVVIP (SEQ ID NO:23)GEVVIPGGVYDYC (SEQ ID NO:24) TNPEPASGKMWIA (SEQ ID NO:25) GKMWIAGDGGNQP(SEQ ID NO:26) DGGNQPARYDDFT (SEQ ID NO:27) RYDDFTFEAGKKY (SEQ ID NO:28)EAGKKYTFTMRRA (SEQ ID NO:29) FTMRRAGMGDGTD (SEQ ID NO:30) MGDGTDMEVEDDS(SEQ ID NO:31) DGTDMEVEDDSPA. (SEQ ID NO:32)

Epitope mapping of the pin-bound peptides was carried out by ELISA asper Chiron Technologies instructions using the human and mouseproteinase antisera at a dilution of 1:1000 in 1% w/v non-fat skim milkpowder in 0.1M phosphate buffered saline, pH 7.4, containing 0.1% v/vTween 20. The bound antibody was detected by incubating the peptide-PINSwith 0.4 mM 3,3′,5,5′-tetramethylbenzidine in 0.1M sodium acetate/citricacid buffer containing 0.004% v/v H₂O₂. Colour development was stoppedby the addition of 2M H₂SO₄. Optical density (O.D.) was measured at 450nm using a BioRad microplate reader model 450.

Antisera from D20, D24, H10 and the protective mouse sera were used toepitope map the N-terminal 148 residues of the PrtR27 adhesin using thePIN-bound overlapping peptides. FIG. 6 clearly shows four regions whichbind antibody and have the sequences: FLLDADHNTFGSVIPATGPLFTGTASS (SEQID NO:1) LYSANFESLIPANADPVVTTQNIIVTG (SEQ ID NO:2) TNPEPASGKMWIAGDGGNQP(SEQ ID NO:4) RYDDFTFEAGKKYTFTMRRAGMGDGTD (SEQ ID NO:5)

The last two epitopes are part of a continuous sequence of the PrtR27adhesin (SEQ ID NO:7) TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.

These protective epitope sequences of the PrtR27 adhesin are also foundin the PrtK39 adhesin and the PrtR44 adhesin of the PrtR-PrtKproteinase-adhesin complex which explains the bands at 44, 39, and 27kDa in the Western blot of the complex using the protective mouse sera.The PrtK39 sequence homologous to the PrtR27 protective epitope isLYSANFEYLIPANADPVVTTQNIVTG (SEQ ID NO:3). The PrtR44 sequence homologousto the Prt27 protective epitope is DDYVFEAGKKYHFLMKKMGSGDGTE. (SEQ IDNO:6)

These sequences incorporated into a peptide construct or peptide proteinconjugate therefore could form the basis of an immunogen to provideprotection against periodontitis.

(ii) Synthesis of Peptide Antigens and Multiple Constructs

The peptides of Table 1 (EP1-EP7) can be synthesized using standard Fmocor tBoc synthesis strategies and multipeptide constructs can besynthesized using the strategies outlined in FIGS. 1-5.

(iii) Preparation of Antibodies

Serum antibodies can be obtained by immunising horses, rabbits, sheep ordairy cows.

Immunizations can be carried out using standard procedures. The initialimmunisation is usually with a mixture of the antigen and Freund'sincomplete adjuvant. The antibodies can be recovered from the animalsserum or milk using standard procedures.

EXAMPLE 2 Methods for Using Antigenic Peptides in DiagnosticImmunoassays

The P. gingivalis peptide antigens described herein can be synthesizedfor use as immunogens in vaccine formulations; and as antigens fordiagnostic assays or for generating P. gingivalis-specific antisera oftherapeutic and/or diagnostic value.

The peptides disclosed in Table 1 can be synthesized individually orchemically-linked using the strategies of FIGS. 1-5. The peptides can besynthesized using one of the several methods of peptide synthesis knownin the art including standard solid phase peptide synthesis usingtertbutyloxycarbonyl amino acids (Mitchell et al., 1978, J. Org. Chem43:2845-2852), using 9-fluorenylmethyloxycarbonyl amino acids on apolyamide support (Dryland et al. 1986. J. Chem. So. Perkin Trans. I,125-137); by pepscan synthesis (Geysen et al. 1987, J. Immunol, Methods03:259; 1984, Proc. Natl. Acad. Sci. USA 81:3998): or by standard liquidphase peptide synthesis. Modification of the peptides or oligopeptides,such as by deletion and substitution of amino acids (and includingextensions and additions to amino acids) and in other ways, may be madeso as to not substantially detract from the immunological properties ofthe peptide or oligopeptide. In particular, the amino acid sequences ofthe antigens described herein, may be altered by replacing one or moreamino acids with functionally equivalent amino acids resulting in analteration which is silent in terms of an observed difference in thephysicochemical behaviour of the peptide, or oligopeptide or chimera.Functionally equivalent amino acids are known in the art as amino acidswhich are related and/or have similar polarity or charge. Thus, an aminoacid sequence which is substantially that of the amino acid sequencesdepicted in the Sequence Listing herein, refers to an amino acidsequence that contains substitutions with functionally equivalent aminoacids without changing the primary biological function of the peptide,oligopeptide or chimera.

Purified synthetic peptides may be used as antigens in immunoassays forthe detection of P. gingivalis-specific antisera present in the bodyfluid of an individual suspected of having an infection caused by P.gingivalis. The detection of antigens or related peptides inimmunoassays includes any immunoassay known in the art including, butnot limited to, radioimmunoassay, enzyme-linked immunosorbent assay(ELISA). “sandwich” assay, precipitin reaction, agglutination assay,fluorescent immunoassay, and chemiluminescence-based immunoassay.

EXAMPLE 3 Synthesis of Protective Epitopes and Testing in a MurineLesion Model

The following peptides representative of the protective epitopes listedin Table 4 were synthesised, conjugated and tested in the murine lesionmodel. TABLE 4 Origin and amino acid sequence of synthesised peptidesAmino acid sequence Abbrevia- Origin (single letter code) tionProtective Peptide Epitopes PrtK39 FLLDADHNTFGSVIPATGPLFTGTASS EP1(531-557) (SEQ ID NO:1) PrtK39 LYSANFESLIPANADPVVTTQNIIVTG EP2 (559-585)(SEQ ID NO:2) PrtK39 TNPEPASGKMWIAGDGGNQP EP4 (601-620) (SEQ ID NO:4)PrtK39 RYDDFTFEAGKKYTFTMRRAGMGDGTD EP5 (622-648) (SEQ ID NO:5) PrtR44DDYVFEAGKKYHFLMKKMGSGDGTE EP6 (604-627) (SEQ ID NO:6)(i) Materials

Unless otherwise stated chemicals were of peptide synthesis grade or itsequivalent. O-Benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), 1hydroxybenzotriazole (HOBt),diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), piperidine,trifluoroacetic acid (TFA) and 9-fluorenylmethoxycarbonyl (Fmoc)protected amino acids were obtained from Auspep Pty Ltd (Melbourne,Australia). Triisopropylsilane (TIPS) and ethanedithiol (EDT) wereobtained from Aldrich (New South Wales, Australia).1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained from SigmaChemical Company (New South Wales, Australia). Phenol and diethyl etherwere obtained from BDH (Poole, UK).

(ii) Solid-Phase Peptide Synthesis

Peptides were synthesised manually or using a 431A ABI peptidesynthesiser. Standard solid-phase peptide synthesis protocols for Fmocchemistry were used throughout. Peptides were assembled as thecarboxyamide form using Fmoc-Pal-Peg-PS resin (PerSeptive BiosystemsInc., Framingham, Mass.). Coupling was accomplished with HBTU/HOBtactivation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA. TheFmoc group was removed by 2% V/V DBU in DMF containing 2% v/vpiperidine. Cleavage of peptides from the resin support was performedusing TFA:phenol:TIFS:EDT:water (92.2:2:2:2) cleavage cocktail for 2.5hours or 4 hours depending on the arginine content of the peptide. Aftercleavage the resin was removed by filtration and the filtrateconcentrated to approximately lm under a stream of nitrogen. After thepeptide products were precipitated in cold ether, they were centrifugedand washed three times. The peptide precipitate was then dissolved in 5to 10 mL of water containing 0.1% v/v TFA and insoluble residue removedby centrifugation.

(iii) Synthesis of S-Acetylmercaptoacetic Acid Peptides

Resins bearing peptides were swollen in DMF and the N-terminal Fmocgroup removed by 2% v/v DBU in DMF containing 2% v/v piperidine.S-Acetylmercaptoacetic acid (SAMA) group was introduced onto theN-terminal amino Croup using 5 equiv of SAMA-OPfp and 5 equiv of HOBt.The reaction was monitored by the trinitrobenzene sulphonic acid (TNBSA)test. When a negative TNBSA test was returned the resin was washed(5×DMF, 3×DCM and 3×diethyl ether). The resin was dried under vacuum andthe SAMA-peptides cleaved from the resin support as described above.

(iv) Peptide Purification:

Purification of synthesized peptides was performed using a Brownlee C18Aquapore ODS column (250×100 mm) installed in a Waters HPLC system.Chromatograms were developed at a flow Tate of 5 mL/min using 0.1% v/vTFA in water (solvent A) and 0.1% v/v TFA in 90% aqueous acetonitrile(solvent B) as the limit buffer, Peptides were eluted with a gradient of10-30% solvent B formed over 40 min. Analytical HPLC was carried outusing a Brownlee C8 Aquapore RP-300 column (220×4.6 mm) installed in aApplied Biosytems HPLC system. Chromatograms were developed usingsolvent A and solvent B at a flow rate of 1 mL/min and a 0-100% lineargradient of solvent B formed over 30 min. Material eluted from thecolumns was detected by determining the absorbance at 214 nm. Peptidefractions were pooled and lyophilised. Peptides were analysed by massspectrometry using a PerSeptive Biosystems Voyager DE MALDI-TOF.

(v) Conjugation of SAMA-Peptides to Diphtheria Toxoid

Diphtheria toxoid (DT) was obtained from CSL Limited, Melbourne.Australia which contained 9 equivalent amino groups per 62 kDa molecule.To a solution containing 10 mg/mL of DT in phosphate-buffered saline(0.1M sodium phosphate, 0.9% NaCl; pH 7.4) was added 0.1 mL of a 1% w/vsolution m-maleimido benzoyl-N-hydroxysuccinimide ester (BS) in DMF.After 30 mins unreacted MBS was removed and MBS modified DT collected bygel filtration using a PD10 column (Pharmacia, NSW, Australia)equilibrated in conjugation buffer (0.1M sodium phosphate, 5 mM EDTA; pH6.0). Purified SAMA-peptide (1.3 μmole) was dissolved in 200 μL 6Mguanidine HCl containing 0.5M Tris; 2 mM EDTA, pH 6 and diluted with 800μL MilliQ water and deprotected in-situ by addition of 25 μL of 2M NH₂OH(40 equiv) dissolved in MilliQ water. The collected MBS-DT wasimmediately reacted with deprotected SAMA-peptide and stirred for onehour at room temperature. The peptide-DT conjugate was separated fromunreacted peptide by gel filtration using a PD10 column equilibrated inPBS pH 7.4 and lyophilised. The reaction was monitored using the Ellmanstest. The conjugation yields of SAMA-peptides to MBS-DT ranged from 34%o to 45% indicating that 3 to 4 peptides were coupled per DT molecule.

(vi) Immunization and Murine Lesion Model Protocols

BALB/c mice 6-8 weeks old were immunised subcutaneously with either 50μg of the peptide-DT conjugate, 50 μg of DT or 25 μg of the PrtR-PrtKproteinase adhesin complex from P. gingivalis strain W50 emulsifiedincomplete Freund's adjuvant (CFA). After 30 days the mice were injectedsubcutaneously with antigen (either 50 μg of the peptide-DT conjugate,50 kg of DT or 25 μg of the PrtR-PrtK proteinase adhesin complex from P.gingivalis strain W50) emulsified in incomplete Freund's adjuvant (IFA)and then bled from the retrobulbar plexus 12 days later. All mice werechallenged with 7.5×10⁹ cells of P. gingivalis (200 μL) by subcutaneousinjection in the abdomen and weighed and lesion size measured over 10days. Lesion sizes are expressed as mm2 and were statistically analysedusing a Kruskal-Wallis one-way ANOVA and Mann-Whitney U-Wilcoxon ranksum test. A Bonferroni correction of type I error was used whencomparing peptide groups with the control (DT) croup.

FIG. 7 shows maximum lesion size that developed for each group.Statistical analysis of the data indicated that mice immunised witheither EP1-DT, EP2-DT, EP4-DT, EP5-DT, EP6-DT and the complex hadsignificantly (p<0.005) smaller lesions than that of the control groupimmunised with DT alone.

The results demonstrate the efficacy of the EP protective peptides whenused as immunogens in preventing challenge with P. gingivalis in themurine lesion model.

EXAMPLE 4 Methods and Compounds for Vaccine Formulations Related toSynthetic Peptide Antigens and Multipeptide Constructs

This embodiment of the present invention is to provide peptide antigensof Table 1 to be used as immunogens in a prophylactic and/or therapeuticvaccine for active immunization to protect against or treat infectionscaused by P. gingivalis. For vaccine purposes, an antigen of P.gingivalis comprising a synthetic peptide construct should beimmunogenic, and induce functional antibodies directed to one or moresurface-exposed epitopes on intact bacteria, wherein the epitope(s) areconserved amongst strains of P. gingivalis.

In one illustration of the invention, the dipeptide EP4-EP5 constructhaving the properties desirable of a vaccine antigen, the dipeptideconstruct can be synthesized using the method described herein in FIGS.1-5.

The synthetic peptide is included as the relevant immunogenic materialin the vaccine formulation, and in therapeutically effective amounts, toinduce an immune response. Many methods are known for the introductionof a vaccine formulation into the human or animal to be vaccinated.These include, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, ocular, intranasal, and oraladministration. The vaccine may further comprise a physiological carriersuch as a solution, a polymer or liposomes; and an adjuvant, or acombination thereof.

Various adjuvants are used in conjunction with vaccine formulations. Theadjuvants aid by modulating the immune response and in attaining a moredurable and higher level of immunity using smaller amounts of vaccineantigen or fewer doses than if the vaccine antigen were administeredalone. Examples of adjuvants include incomplete Freund's adjuvant (ISA),Adjuvant 65 (containing peanut oil, mannide monooleate and aluminummonostrearate), oil emulsions, Ribi adjuvant, the pluronic polyols,polyamines, Avridine, Quil A, saponin, MPL, QS-21, and mineral gels suchas aluminum hydroxide, aluminum phosphate, etc.

Another embodiment of this mode of the invention involves the productionof antigen-specific amino acid sequences as a hapten, i.e. a moleculewhich cannot by itself elicit an immune response. In such case, the hapten may be covalently bound to a carrier or other immunogenic moleculewhich will confer immunogenicity to the coupled hapten when exposed tothe immune system. Thus, such a antigen-specific hap ten linked to acarrier molecule may be the immunogen in a vaccine formulation.

As an alternative to active immunization, immunization may be passive,i.e. immunization comprising administration of purified immunoglobulincontaining antibody against synthetic peptides.

EXAMPLE 5

The following is a proposed toothpaste formulation containinganti-peptide antibodies. Ingredient % w/w Dicalcium phosphate dihydrate50.0 Glycerol 20.0 Sodium carboxymethyl cellulose 1.0 Sodium laurylsulphate 1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin0.1 Chlorhexidine gluconate 0.01 Dextranase 0.01 Goat serum containinganti-peptide Abs 0.2 Water balance

EXAMPLE 6

The following is a proposed toothpaste formulation. Ingredient % w/wDicalcium phosphate dihydrate 50.0 Sorbitol 10.0 Glycerol 10.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Bovine serum containing anti-peptide Abs 0.2 Water balance

EXAMPLE 7

The following is a proposed toothpaste formulation. Ingredient % w/wDicalcium phosphate dihydrate 50.0 Sorbitol 10.0 Glycerol 10.0 Sodiumcarboxymethyl cellulose 1.0 Lauroyl diethanolarmide 1.0 Sucrosemonolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 01 Dextranase 0.01Bovine milk Ig containing anti-peptide Abs 0.1 Water balance

EXAMPLE 8

The following is a proposed toothpaste formulation. Ingredient % w/wSorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0 Gantrez 19.0Water (deionised) 2.69 Sodium Monofluorophosphate 0.76 Sodium saccharine0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavour oil 0.95anti-peptide mouse monoclonal 0.3 sodium lauryl sulphate 2.00

The following is a proposed liquid toothpaste formulation. Ingredient %w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0 Flavour 1.0Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidinegluconate 0.01 Ethanol 3.0 Equine Ig containing anti-peptide Ab 0.2Linolic acid 0.05 Water balance

EXAMPLE 10

The following is a proposed mouthwash formulation. Ingredient % w/wEthanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate0.3 Chlorhexidine gluconate 0.01 Lauroyl diethanolamide 0.3 Rabbit Igcontaining anti-peptide-Ab 0.2 Water balance

EXAMPLE 11

The following is a proposed mouthwash formulation. Ingredient % w/wGantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 Mouse anti-peptide monoclonal 0.3 Water balance

EXAMPLE 12

The following is a proposed lozenge formulation. Ingredient % w/w Sugar75-80 Corn syrup  1-20 Flavour oil 1-2 NaF 0.01-0.05 Mouse anti-peptidemonoclonal 0.3 Mg stearate 1-5 Water balance

EXAMPLE 13

The following is a proposed gingival massage cream formulation.Ingredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stearylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 Mouse anti-peptidemonoclonal 0.3 Water balance

EXAMPLE 14

The following is a proposed chewing gum formulation. Ingredient % w/wGum base 30.0 Calcium carbonate 2.0 Crystalline sorbitol 53.0 Glycerine0.5 Flavour oil 0.1 Mouse anti-peptide monoclonals 0.3 Water balance

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

-   Alexander, J., Sidney. J., Southwood, S., et al (1994). Development    of high potentcy universal DR-restricted helper epitopes by    modification of high affinity DR-blocking peptides. Immunity 1:    751-761.-   Canne, L. E. Ferre-D'Amare, A. R., Burley, S. K. and Kent, S. B. H.    (1995). Total chemical synthesis of a unique transcription    factor-related protein; cMyc-Max. J. A. Chem. Soc. 117: 2998-3001.-   Druland, et. al. (1986). J. Chem. Soc. Perkin Trans. 1: 125-137.-   Duncan, R., and Kopececk, J. (1980). Degradation of side chains of    N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal enzymes.    Biochem. Biophys. Res. Commun. 94; 284-290.-   Geysen, H. M., Meleon, R. H., and Barteling, S. J. (1984). Use of    peptide synthesis to probe viral antigens for epitopes to a    resolution of a single amino acid. Proc. Natl. Acad. Sci. USA. 81:    3998.-   Geysen, H. M. Rodda, S. J., Mason, T. J., et al. (1987). Strategies    for epitope mapping using peptide synthesis. J. Immunol. Methods.    102: 259.-   Hammer, J., Valsasnini, P., Tolba, K., Bolin, D., Higelin, J.,    Takacs, B., and Sinigaglia, F. (1993). Promiscuous and    allele-specific anchors in HLA-DR-binding peptides. Cell 74:    197-203.-   Kaumaya. P. T. P., Kobs-Conrad, S., and DiGeorge, A. M. (1994).    Synthetic peptide vaccines: Misconceptions and problems, strategies    and prospects Innovation and Perspectives in Solid Phase    Synthesis. R. Epton. Kingswinford, Mayflower: 279-292.-   Kesavalu, L., Ebersole, J. L., Machen, R. L., Holt, S. C. (1992).    Porphyromonas gingivalis virulence in mice: induction of immunity to    bacterial components. Infect. Immun. 60: 1455-1464-   Liu, C. F. a. T., J.P. (1994). Peptide ligation strategy without use    of protectecing groups. Proc. Natl. Acad. Sci. USA 91: 6584-6588.-   Lu, Y. A. Clavijo, P., Galantino, M., Shen, Z. Y., and Tam, J.P.    (1991). Chemically unambiguous peptide immunogen: Preparation,    orientation and antigenicity of purified peptide conjugated to the    multiple antigen peptide system. Mol. Immunol. 28(6): 623-630.-   Mitchell., e. a. (1978). J. Org. Chem. 43: 2845-2852.-   O'Brien-Simpson, N. M., Ede, N. J., Brown, L. E., Swan, J., and    Jackson, D. C. (1997). Polymerisation of unprotected synthetic    peptides: a view towards a synthetic peptide vaccines. J. Am. Chem.    Soc. 117(6).-   O'Sullivan, D., Arrhenius, T., Sidney, J., et al (1991). On the    interaction of promiscuous antigenic peptides with different DR    alleles. Identification of common structural motifs. J. Immunol    147(8): 2663-2669.-   Rose, K. (1994). Facile synthesis of homogeneous artificial    proteins. J. Am. Chem. Soc. 116: 30-33.-   Rose, J., Zeng, W., Regamey, P. O., Chernusheivich, I. V.,    Standing, K. G. and Gaertner, H. F. (1996). Natural peptides as    building blocks for the synthesis of large protein-like molecules    with hydrazone and oxime linkages. Bioconjugate Chem. 7(5): 552-556.-   Shao, J., and Tam, J. P. (1995). J. Am. Chem. Soc. 117: 3893-3899.-   Spetzler, J. C. a. T., J. P. (1994). A general approach for the    synthesis of branched peptides for synthetic vaccines: Synthesis of    multiple antigen peptides using unprotected segments. Innovation and    Perspectives in Solid Phase Synthesis. R. Epton. Kingswinford,    Mayflower: 293-300.-   van Noort, J. M., and van der Drift, A. C. M. (1989). The    selectivity of cathepsin D suggeste an involvement of the enzyme in    the generation of T-cell epitopes. J. Biol. Chem: 264(24):    14159-14164.

1. A composition for use in raising an immune response againstPorphyromonas gingivalis, the composition comprising a suitable adjuvantand/or acceptable carrier or excipient and at least one peptide of notmore than 50 amino acids which peptide includes at least one P.gingivalis epitope, or multimers of said peptide, the at least one P.gingivalis epitope being selected from the epitopes included within apeptide selected from the group consisting of: (SEQ ID NO:1)FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ ID NO:2) LYSANFESLIPANADPVVTTQNIIVTG,(SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4)TNPEPASGKMWIAGDGGNQP (SEQ ID NO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ IDNO:6) DDYVFEAGKKYHFLMKKMGSGDGTE and (SEQ ID NO:7)TNPEPASGKMWLAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


2. A composition as claimed in claim 1 in which the at least one peptidecomprises a sequence selected from the group consisting of:NTFGSVIPATGPL, (SEQ ID NO:8) LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:2)LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:3) PASGKMWLAGDG, (SEQ ID NO:9)EAGKKYTFTMRRA (SEQ ID NO:10) and EAGKKYHFLMKKM. (SEQ ID NO:11)


3. A composition as claimed in claim 1 in which the at least one peptidecomprises a sequence selected from the group consisting of: (SEQ IDNO:1) FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ ID NO:2)LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG,(SEQ ID NO:4) TNPEPASGKMWIAGDGGNQP, (SEQ ID NO:5)RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ ID NO:6) DDYVFEAGKKYHFLMKKMGSGDGTE and(SEQ ID NO:7) TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


4. A composition as claimed in claim 1 in which the at least one peptideis selected from the group consisting of: (SEQ ID NO:1)FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ ID NO:2) LYSANFESLIPANADPVVTTQNIIVTG,(SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4)TNPEPASGKMWIAGDGGNQP, (SEQ ID NO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ IDNO:6) DDYVFEAGKKYHFLMKKMGSGDGTE and (SEQ ID NO:7)TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


5. A composition as claimed in claim 1 which comprises more than onepeptide.
 6. A composition as claimed in claim 5 in which the peptidesare in multimeric form(s).
 7. A composition as claimed in claim 6 inwhich the multimer(s) comprise different peptides.
 8. A peptide havingno more than 50 amino acids which peptide comprises at least one P.gingivalis epitope, the at least one P. gingivalis epitope beingselected from the epitopes included within a peptide selected from thegroup consisting of: (SEQ ID NO:1) FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ IDNO:2) LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:3)LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4) TNPEPASGKMWLAGDGGNQP, (SEQ IDNO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ ID NO:6)DDYVFEAGKKYHFLMKKMGSGDGTE and (SEQ ID NO:7)TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


9. A peptide as claimed in claim 8 comprising at least one sequenceselected from the group consisting of: NTFGSVIPATGPL, (SEQ ID NO:8)LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:2) LYSANFEYLIPANADPVVTTQNIIVTG,(SEQ ID NO:3) PASGKMWLAGDG, (SEQ ID NO:9), EAGKKYTFTFMRRA (SEQ ID NO:10)and EAGKKYHFLMKKM. (SEQ ID NO:11)


10. A peptide as claimed in claim 8 comprising at least one sequenceselected from the group consisting of: (SEQ ID NO:1)FLLDADHNTFGSVIPATGPLFTGTASS, (SEQ ID NO:2) LYSANFESLIPANADPVVTTQNIIVTG,(SEQ ID NO:3) LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4)TNPEPASGKMWIAGDGGNQP, (SEQ ID NO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ IDNO:6) DDYVFEAGKKYHFLMKKMGSGDGTE and (SEQ ID NO:7)TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


11. A peptide as claimed in claim 8 in which the peptide is selectedfrom the group consisting of: (SEQ ID NO:1) FLLDADHNTFGSVIPATGPLFTGTASS,(SEQ ID NO:2) LYSANFESLIPANADPVVTTQNIIVTG, (SEQ ID NO:3)LYSANFEYLIPANADPVVTTQNIIVTG, (SEQ ID NO:4) TNPEPASGKMWIAGDGGNQP, (SEQ IDNO:5) RYDDFTFEAGKKYTFTMRRAGMGDGTD, (SEQ ID NO:6)DYVFEAGKKYHFLMKKMGSGDGTE and (SEQ ID NO:7)TNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTD.


12. Use of a peptide as claimed in claim 8 as an antigen in a diagnostictest.
 13. An antibody specifically directed against a composition asclamed in claim 1 or a peptide as defined above.
 14. An antibody asclaimed in claim 13 which is a monoclonal antibody.
 15. Use of anantibody as claimed in claim 13 in a diagnostic test.
 16. A compositioncomprising an antibody as claimed in claim 13 and a pharmaceuticallyacceptable carrier or diluent.
 17. A method of reducing the prospect ofP. gingivalis infection in an individual and/or severity of disease, themethod comprising administering to the individual an amount of acomposition as claimed in claim 1 effective to induce an immune responsein the individual directed against P. Gingivalis.
 18. A method ofreducing the prospect of P. gingivalis infection in an individual and/orseverity of disease, the method comprising administering to theindividual an effective amount of a composition as claimed in 16.